The present study was undertaken to evaluate the effect of trisaccharides supplementation in glycerol-free tris (GFT) for the cryopreservation of dog spermatozoa. In the first experiment (E1), dog spermatozoa were resuspended with 50, 75, 100 or 125 mM of raffinose, melezitose or maltotriose and cooled at 4 ℃ for 10 min. To determine the effect of different cooling time, the spermatozoa resuspended with 100 mM of raffinose, melezitose or maltotriose were cooled during 10, 20, 30 or 40 min at 4 ℃ (second experiment; E2). The straws were then aligned horizontally for 10 min on the rack and then plunged into LN2. In the third experiment (E3), to determine the effect of different vapor freezing time, the spermatozoa resuspended with 100 mM raffinose were cooled at 4 ℃ for 20 min and frozen in LN2 for 5, 10, 15 or 20 min and then plunged into LN2. In the fourth experiment (E4), to compare different freezing methods [cooling plus vapor freezing (CV), cooling plus step-down freezing (CS) and direct step-down freezing (SD)], the spermatozoa resuspended with 100 mM raffinose were cooled for 20 min and frozen in LN2 vapor for 5 min in case of CV method. In case of CS method, spermatozoa were cooled for 20 min at 4℃ and then frozen by the step-down freezing method. The straws were then aligned horizontally at 18, 15, 5, and 2 cm respectively from the surface of LN2 for 1, 1, 1.4, and 5 min, respectively in an L shaped straw holder and then plunged into LN2. For SD method, the straws were directly aligned horizontally at the same levels as CS from the surface of LN2 for 1, 1, 1.9, and 5 min, respectively and then plunged into LN2. After thawing at 37℃ for 25 sec, the spermatozoa were then incubated for 30 min in the freezing extender (E1) or in the 50 mM sucrose supplemented GFT (E2, E3, and E4) at 24℃. Following post-thaw incubation, sperm progressive motility and viability were assessed in E1, E2, E3, and E4. In addition, acrosome integrity, and gene expression related to apoptosis (BAX, BCL2, and Caspase10) and sperm motility (SMCP) were evaluated in E4. The results demonstrated that, in E1, using 75 mM trisaccharides resulted in significantly (p<0.05) higher sperm motility in all sugar groups. Using 100 mM melezitose significantly (p<0.05) improved the post-thaw viability than the 100 mM raffinose. The viability in 100 mM maltotriose was similar with 100 mM raffinose and melezitose group. In E2, the different cooling time has no significant effect on post-thaw sperm progressive motility in all the sugar types. In addition, the viability was variable among the different groups. In E3, liquid nitrogen vapor freezing for 5 min resulted in improved motility and viability. The sperm progressive motility was significantly (p<0.05) higher in CV and SD group compared to CS group and the sperm viability was significantly (p<0.05) higher in CV group compared to the other groups in E4. However, the acrosomal integrity of spermatozoa in the group CV was significantly (p<0.05) higher than the group CS and SD. In addition, the expression of SMCP gene was significantly (p<0.05) higher in the CV group than the CS group. In contrast, the expression of Caspase10 significantly (p<0.05) lower in the group CV and SD than the group CS. Furthermore, the ratio of gene expression of BAX and BCL2 was significantly (p<0.05) lower in the group CV than the group CS. Therefore, cryopreservation of dog spermatozoa in 100 mM of raffinose supplemented GFT cooled for 20 min and vapor freezing for 5 min provides better progressive sperm motility, viability, and acrosome integrity with higher expression of SMCP gene and lower expression of caspase10 and BAX/BCL2 ratio following post-thaw incubation in 50 mM sucrose supplemented GFT for 30 min at 24℃.

To preserve the superior genetic resources and restore the endangered species, Somatic cell nuclear transfer (SCNT) has been used widely. In Korea, the research of dog cloning has made outstanding achievements including the production of the world`s first cloned dog. Sapsaree (Sapsalgae), the representative dog of Gyeongsan-si was designated as a Korea natural monument (No. 368). This male dog used in this study has azoospermia due to unknown cause. In this study, the aim was to confirm the cause of infertility in the cell donor dog and to evaluate the reproduction potential of dog cloning using infertile male dog by SCNT. First, to confirm the infertility of the cell donor dog, the reproductive history and the testis were evaluated. The breeding histology was not recorded in individual document. In histopathology, the Sertoli cell tumor was confirmed in biopsy of the cell donor dog after death. But, these tumors are predominantly in older dogs. Second, we produced the cloned dogs with the somatic cells of the infertile dog and the appearance was similar with the cell donor dog. Also, microsatellite analysis confirmed the genetic relationship between the cell donor and clone dogs. Third, the potential breeding capacity of the cloned dog was confirmed. In T4 assay, the normal dog (same age with cloned dogs), cell donor dog, and cloned dogs was investigated. The cell donor dog with azoospermia had very low T4 level, and cloned dogs showed higher level of T4 than normal dogs. In CASA, There was no significant difference in sperm motor ability between normal dogs and cloned dogs. As a result, cloned dogs produced by SCNT had no problem regarding the reproductive function of the testis. In AI experiment, the semen of clone dogs was used to fertilize a natural female bitch and was diagnosed pregnancy by ultrasonography. In total, 7 puppies were born by normal delivery (male: 3, female: 4). In conclusion, this study confirmed that the reproduction problem of non-genetic infertility can generate a normal descendant by SCNT. Also, the first successful research to restore infertile dogs was completed. Furthermore, SCNT would be useful for the restoration of endangered species and application of superior traits.

Porcine Reproductive and Respiratory Syndrome (PRRS) is the most economically important disease in swine in North America, Europe and Asia. PRRS is caused via infection of the pulmonary alveolar macrophages (PAMs) with the PRRS virus (PRRSV) causing respiratory illness and high fever in young growing pigs that predisposes them to secondary bacterial infections. PRRSV also causes severe reproductive failure in sows and boars. Although research is ongoing, PRRSV continues to elude a successful vaccine. In 2014, piglets were born with a gene edit in exon 7 of the Cluster of differentiation 163 (CD163) gene introduced by using the CRISPR/Cas9 site-directed nucleases system. The resulting litters of pigs were either challenged with multiple PRRSV isolates at 3 weeks of age or bred at maturity for a challenge with pregnant sows. The challenges demonstrated that the pigs were completely resistant to infectivity to both Type 1 and 2 isolates as measured by clinical signs, viremia, antibody response and lung histopathology. In a follow-up study, pregnant CD163-/- pigs were also challenged with PRRSV to determine if absence of CD163 in the dam should be sufficient to protect the CD163+/- fetuses that have functional CD163 protein. The wild-type sow and fetuses were actively infected with the PRRSV and one sow aborted. The CD163-/- sows carrying both the CD163-/- and CD163+/- fetuses were all negative for PRRSV nucleic acid and showed no sign of fetal or placental failure. The results of this study clearly demonstrate that the absence of CD163 in the sow is sufficient to protect a PRRSV-susceptible CD163+/- fetus. Gene editing of CD163 in pigs, via CRISPR/Cas9, successfully blocked PRRSV infectivity in young growing pigs and pregnant sows and their fetuses. This is a great example of the potential of utilizing gene editing to improve animal agriculture.

Bemisia tabaci (Gennadius) is an important polyphagous pest and transmits plant pathogenic virus to numerous agriculture crops. We compared the efficacy of Beauveria bassiana ARP14 strain with commercialized GHA strain against 2nd instar and 4th instar of B. tabaci. The nymphs were exposed in 1×108 conidia/mL by dipping method. There was no difference in mycosis rate between the two strains in both 2nd and 4th instar. After 72 h, mycosis rate of 2nd instar was 100.0% in B. bassiana ARP14 strain followed by 97.4% in B. bassiana GHA strain. In 4th instar, B. bassiana ARP14 strain caused 100.0% while B. bassiana GHA strain caused 93.3%.

Drosophila suzukii (Matsumura) is one of the most destructive pest of thin-skinned fruits such as blueberry, strawberry, raspberry, and cherry. We tested effect of fruit damage on the infestation of D. suzukii by using partially peeled blueberry fruits in assays of behavioral choice and development. In choice test, D. suzukii preferred partially peeled blueberry fruit than normal. But in development test, we did not found difference in adult emergence rate between normal and partially peeled blueberry fruit.

Geminiviruses are plant-infecting viruses with monopartite or bipartite single-stranded circular DNA genomes. They are known to be mediated by insects such as whiteflies, treehoppers, leafhoppers or aphids and can cause devastating plant diseases in a wide range of economically significant crops worldwide. In Korea, occurrence of geminiviruses were reported officially after the 2000s. Although Honeysuckle yellow vein virus (HYVV) and Sweet potato leaf curl virus (SPLCV) were identified from honeysuckle and sweet potato in 2004 and 2006 respectively, these viruses did not spread and cause much concern for geminiviruses. In 2008, Tomato yellow leaf curl virus (TYLCV) that has caused severe tomato production loss in many subtropical and tropical countries was first reported in tomato plants cultivated in Tongyeong. TYLCV rapidly spread through the country and has been continuously reported from tomato cultivating areas in Korea. In addition to TYLCV, Tobacco leaf curl virus and Sweet potato golden vein associated virus have occurred. In recent years, new geminiviruses including Papaya leaf curl Guangdong virus (PaLCGdV) and Euphorbia leaf curl virus (EuLCV) have been introduced. Newly emerging geminiviruses from tropical and subtropical countries are due to increased demand for various fruits and vegetables, and climate change. In addition, there are reports that some geminiviruses including TYLCV can be transmitted by infected seeds. Therefore it is important to study on epidemiology of virus introduction and spread among the countries and within country.

Frequent surveys and monitoring were conducted in the Southern part of Bangladesh to detect and identify the plant pathogenic virus that infecting agriculturally important vegetables during 2017-2018. A total of 28 fields of the survey area were closely monitored. The findings indicated that 21.94% of the plants developed typical virus disease like symptomps in the field. However, 28.21% infected plants were found in Patuakhali followed by Satkhira (23.11%), Khulna (19.33%) and Barguna (17.12%). The symptoms were mosaic, vein clearing, chlorosis, curling and ringspot. Twenty samples from the collections were randomly chosen on the basis of symptoms and subjected to Enzyme-linked Immunosorbent Assay (ELISA) with the antiserum and symptomalogy were used for detection. Eight viruses namely Cucumber mosaic virus (CMV), Okra yellow vein clearing mosaic virus (OYVCMV), Mungbean yellow mosaic virus (MYMV), Tomato yellow leaf curl virus (TYLCV), Pepper mottle virus (PMV), Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV) were detected on cucumber, okra, brinjal, mungbean, tomato, pepper, papaya, watermelon and pumpkin respectively.

We investigated the detoxification strategies of Helicoverpa armigera and Heliothis virescens, which allow them to feed successfully on cotton plants that produce toxic gossypol as a chemical defense compound. First, we tested CYP6AE14, a proposed candidate enzyme for gossypol detoxification, for its ability to detoxify gossypol. In incubation assays with gossypol and heterologously expressed CYP6AE14 no metabolites were detected. Our data show that CYP6AE14 is not directly involved in gossypol metabolism, at least under the assay conditions tested, but rather takes part in the general stress response of the herbivores to plant toxins. Second, we discovered that H. armigera and H. virescens excrete a large proportion (50%) of unmetabolized gossypol in the feces, but additionally metabolize gossypol by glycosylation. Analysis of larval feces revealed three monoglycosylated and up to five diglycosylated gossypol isomers when larvae fed on gossypol-supplemented diet. Based on their expression patterns we selected H. armigera candidate UGT genes and functionally expressed the respective proteins in insect cells. In enzymatic assays, we showed that UGT41B3 and UGT40D1 are capable of glycosylating gossypol mainly to a diglycosylated gossypol isomer that is characteristic for H. armigera and is absent in H. virescens feces. We offer novel insights into the detoxification mechanism of the plant defensive toxin, gossypol, by two generalist herbivores.

Cold, salt and heat are the most critical factors that restrict full genetic potential, growth and development of crops globally. However, clarification of genes expression and regulation is a fundamental approach to understanding the adaptive response of plants under unfavorable environments. In this study, we applied an annealing control primer (ACP) based on the GeneFishing approach to identify differentially expressed genes (DEGs) in Italian ryegrass (cv. Kowinearly) leaves under cold, salt and heat stresses. Two-week-old seedlings were exposed to cold (4°C), salt (NaCl 200 mM) and heat (42°C) treatments for six hours. A total 8 differentially expressed genes were isolated from ryegrass leaves. These genes were sequenced then identified and validated using the National Center for Biotechnology Information (NCBI) database. We identified several promising genes encoding light harvesting chlorophyll a/b binding protein, alpha-glactosidase b, chromosome 3B, elongation factor 1-alpha, FLbaf106f03, Lolium multiflorum plastid, complete genome, translation initiation factor SUI1, and glyceraldehyde-3-phosphate dehydrogenase. These genes were potentially involved in photosynthesis, plant development, protein synthesis and abiotic stress tolerance in plants. However, this study provides new insight regarding molecular information about several genes in response to multiple abiotic stresses. Additionally, these genes may be useful for enhancement of abiotic stress tolerance in fodder crops as well a crop improvement under unfavorable environmental conditions.

Plant genetic resources are fundamental materials for crop improvement to enhance productivity and an insurance against unforeseen threats to agricultural production. Continuous advancement in crop improvement depends on discovery of new sources of genetic variation, accurate identification of lines with favorable traits, and their efficient and judicious use. Core collections (~10% of the entire collection) and mini core collections (~10% of the core or ~1% of the entire collection) have been suggested as a gateway to enhance utilization of germplasm. Using passport data, characterization and evaluation data, core and/or mini core collections have been developed in chickpea, groundnut, pigeonpea, pearl millet, sorghum, finger millet and foxtail millet at ICRISAT, Patancheru, India. Evaluation of these subsets has resulted in identification of new sources of genotypic variation. The concept and process of developing mini core collections has been recognized worldwide as an “International Public Good” (IPG). Many national programs have shown immense interest in evaluating mini core collections for identification of new sources of variation for use in crop improvement programs. To date, 84 sets of mini core of chickpea, groundnut, pigeonpea, sorghum, pearl millet, foxtail millet and finger millet have been supplied to researchers in 13 countries. Feedback revealed that researchers in national programs were able to identify new sources of variation for favorable traits, such as early maturity, resistance to pests and diseases, large seed size, and high grain yield. Seeds of mini core collections could be available to researchers globally for research and training purpose.